In recent years, particularly in the post-genome era, sugar chain-containing polymers existing as intracellular and extracellular membrane constituents and extracellular molecules in multi-cellular biological organisms, particularly higher biological organisms have drawn attention in relation with the functions thereof in such biological organisms. One typical example of the sugar chain-containing polymers is glycoprotein.
With some exceptions, most of cellular surface membranes and serum proteins in many animals primarily including humans are glycoproteins. For example, antibodies, receptors, hormones and enzymes are not simple proteins but are commonly glycoproteins. The functions of such glycoproteins in biological organisms have traditionally been described from the standpoint of protein structure alone. Since the fact was found that the specificities of ABO (H) type-blood group antigens are determined on the basis of the subtle difference in their sugar chain moieties, however, the roles of sugar chains in glycoproteins as signals have increasingly been focused in relation with various discrimination phenomena required for the establishment and retention of multi-cellular biological organisms.
Specifically, a protein is expressed on the basis of a genetic information and is then glycosylated (added with a sugar chain), to give a biological selectivity extremely specific to the function of the protein itself to the resulting glycoprotein. Glycoproteins are widely distributed in tissues and organisms of animals and plants. Due to the significance of the sugar chains in glycoproteins, diverse and enormous research works have been done and accumulated, for example research works about tumor specific antigens derived from sugar chains. Concurrently, the analysis of sugar chain structures (sequences and steric structures thereof) has made a great progress.
Sugar chains of glycoproteins are broadly classified in N-glycoside linkage type (Asn linkage type) where the sugar chains are linked to L-asparagine as an amino acid residue composing polypeptides and in O-glycoside linkage type (O linkage type) where the sugar chains are linked to L-serine or L-threonine. Among them, sugar chains of O linkage type are found in a wide range including for example various mucous proteins, serum proteins and membrane proteins. Typically, such sugar chains of O linkage type form α-O-glycoside linkage via a nucleophilic reaction using N-acetyl-D-galactosamine as the donor and the alcoholic hydroxyl group of L-serine or L-threonine as the acceptor.
For research works and analyses about the sugar chains of glycoproteins or about lectin drawing attention as a functional protein specifically recognizing the sugar chains of glycoproteins, for example, glycoprotein samples should be prepared at an amount of about several micrograms or more. Because the intended glycoproteins exist at an extremely trace amount in the order of nanogram/milliliter in tissues and cells of animals and plants, generally, the preparation of needed amounts of glycoprotein samples is laborious.
Therefore, research works about the chemical synthesis of glycoproteins of Asn linkage type and O linkage type have been done intensively worldwide. Setting research works about the chemical synthesis of glycoproteins of Asn linkage type aside, various approaches for the chemical synthesis of the glycoproteins of O linkage type have been proposed in terms of O-glycoside linkage very common in the field of sugar chemistry according to techniques developed for the glycosylation of sugar residues together.
The chemical synthesis of glycoproteins of O linkage type involves a very difficult problem as to how the glycosylation between galactosamine at the reducing end and the alcoholic hydroxyl group of amino acid can be facilitated in an α-selective manner. In the glycosylation of N-acetyl-D-galactosamine, in other words, the N-acetylamino group at position 2 interferes with the glycosylation through the neighboring group participation. Accordingly, β-glycosylation also occurs at a considerable ratio, disadvantageously.
Paulsen, et al. propose a process using a 2-azide derivative so as to cope with the disadvantage. As shown in FIG. 1, the process includes the stereo-specific cleavage of 1,6;2,3-dianhydrosugar (i) with azide anion to prepare 2-azide (ii), the conversion of the resulting 2-azide to synthetically prepare α-bromide (iv), and the treatment of the resulting α-bromide with tetraethylammonium chloride to prepare β-chloride (v), to thereby prepare α-glycoside under the Koenigs-Knorr reaction conditions.
A simple synthetic process of 2-azide sugar was then developed by Lemieux and Ratcliffe. As shown in FIG. 2, Ferrari and Pavia converted the compound (x) obtained by the synthetic process to β-chloride (xi), which was then condensed with an L-serine derivative, using a mercury salt as a promoter, to obtain α-glycoside (xii) at a yield of 66%.
Furthermore, Paulsen, et al. utilized silver perchlorate and silver carbonate (at a ratio of 1:10) as the promoter in a non-polar mixture solvent of methylene chloride/toluene so as to suppress unnecessary anomerization of the compound (xi) in FIG. 2. The process produced a fruitful result of the yield of 85% and the α selectivity/β selectivity ratio of 19:1.
The various synthetic processes described above, particularly the process of Paulsen, et al. using silver perchlorate and silver carbonate as the promoter produced a very great result from the standpoint of the issue of the (α-selective glycosylation. However, the processes have common disadvantages of poor practical wide applicability in view of donor preparation stage and tough reaction control.